Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Author:

Wu Shih-Lu,Li Chia-Cheng,Chen Jaw-Chyun,Chen Yi-Jin,Lin Ching-Ting,Ho Tin-Yun,Hsiang Chien-Yun

Abstract

Abstract Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena.

Publisher

Springer Science and Business Media LLC

Subject

Pharmacology (medical),Biochemistry (medical),Cell Biology,Clinical Biochemistry,Molecular Biology,General Medicine,Endocrinology, Diabetes and Metabolism

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